Catharanthine with vindoline

More recently, Kutney and co-workers (220) have investigated whether the same dihydropyridinium intermediate 109 is involved in the enzymatic conversion of catharanthine (4) and vindoline (3) to anhydrovinblastine (8) as is involved in the chemical conversion. Use of a cell-free preparation from a 5-day culture of the AC3 cell line gave 18% of the bisindole alkaloids leurosine (11), Catharine (10), vinamidine (25), and hydroxy-vinamidine (110), with 10 predominating. When the incubations were carried out for only 5-10 min, the dihydropyridinium intermediate was detected followed by conversion to the other bisindole alkaloids, with FAD and MnClj required as cofactors. Clearly a multienzyme complex is present in the supernatant, but further purification led to substantial loss of enzymatic activity. The chemical formation of anhydrovinblastine (3) is carried out with catharanthine A-oxide (107), but when this compound was used in the enzyme preparation described, no condensation with vindoline (3) occurred to give bisindole alkaloids. This has led Kutney and co-workers to suggest (220) that the A-oxide 108 is not an intermediate in the biosynthetic pathway, but rather that a 7-hydroperoxyindolenine... 

In another search for an alternative to Potier s modified Polonovski reaction of catharanthine A-oxide (45), it has now been found that anhy-drovinblastine (42) can be generated directly, in 77% yield, from a reaction of catharanthine and vindoline in 0.01 N acid, promoted by ionized ferric salts, followed by reduction with sodium borohydride (Scheme 30) (Wl). Remarkably, the cation radical 106 generated by Fe(III), in accord with other simple amine oxidations by Lindsay Smith and Mead (102), resulted in isoquinuclidine fragmentation and coupling to vindoline at 0°C, without the conformational inversion observed in the modified Polonovski reaction at that temperature (see Scheme 15). Other metal oxidants or ligand-bound Fe(lll) did not promote the coupling reaction. It will be of interest to see if the overwhelming competition of C-5-C-6 bond... 

The critical dependence of the stereochemical and regiochemical course of the modified Polonovski reaction on the oxygen functionality in the catharanthine derivative has been well exemplified in recent synthetic studies. Indeed, in the reaction that ultimately provided the first synthesis of anhydrovinblastine, a minor product proved to be the result of an alternative fragmentation of the catharanthine Nb-oxide derivative in which the 5,6-bond was cleaved [->(266)] and subsequent coupling of vindoline occurred at position 6, with formation of the dimeric species (267).159 When an attempt was made to couple the N-oxide of the lactone (238) with vindoline under Polonovski conditions, this type of coupling occurred exclusively, and the products were the lactone (268) (major product)163-165, the... 

The dimerization of catharanthine and vindoline is believed to proceed via the formation of an unininm intermediate with catharanthine (Fig. 2e). This unininm intermediate is reduced to form anhydrovinblastine, a naturally occurring compound in C. roseus plants (115). In support of this mechanism, anhydrovinblastine is incorporated into vinblastine and vincristine in feeding studies (116-119). 

An important landmark in the elucidation of the structure of vinblastine (169), C46H58O9N4, was the discovery that the i.r. spectrum was largely super-imposable on the addition spectrum of two monomeric Vinca alkaloids, namely vindoline (171) and catharanthine (172). The structures of these two alkaloids, vindoline with a 6-methoxyindoline chromophore, catharanthine with an indole chromophore unsubstituted on nitrogen, were at that time unknown. 

In 1975, Potier and collaborators proposed that, in planta, the dimeric vinblastine type alkaloids resulted from the coupling of catharanthine and vindoline and, in light of this hypothesis, they reported for the first time the chemical synthesis of a dimer with the natural configuration through a modified Polonovski reaction [18, 19]. This reaction resulted in the formation of an iminium dimer which, after reduction with NaBH4, yielded a-3 ,4 -anhydrovinblastine, Fig. (2), later proved to be the first dimeric biosynthetic precursor of vinblastine in the plant. The group of Potier investigated possible modifications of anhydrovinblastine and produced vinorelbine, Fig. (1), which was the first active derivative with an altered cleavamine (catharanthine) moiety [20, 21]. 

In face of the structural similarities unraveled during the 1960s of vindoline and catharanthine with the dimeric alkaloids, and due to their great abundance in the plant, these two compounds were immediately considered the most likely monomeric precursors of the Vinca alkaloids, although the cleavamine moiety of vinblastine presented some differences from catharanthine, namely a fragmentation of the C5-C18 bond, Fig. (2). 

The chemical coupling of catharanthine and vindoline to yield anhydrovinblastine led to the obvious hypothesis that this compound might also be the first product of dimerization in the plant, and the dimeric precursor of vinblastine and vincristine. For three years it was not possible to find anhydrovinblastine in the plant, until Scott et al. in 1978 [115], by modifying the established methods for extraction and purification of alkaloids, isolated anhydrovinblastine from C. roseus plants, with incorporation of radiolabelled catharanthine and vindoline, thus proving that anhydrovinblastine was actually a natural product. 

At this point, anhydrovinblastine had been proved to actually be a major alkaloid present in C. roseus leaves [106, 107] representing together with catharanthine and vindoline the three major alkaloids of the plant. This indicated the presence of high in vivo anhydrovinblastine synthase activity in leaves and that this was the appropriate biological material to search for the enzyme. Work with leaves started at the laboratory of Prof. Frank DiCosmo from the University of Toronto, Canada, and has mostly been developed in our labs, at the University of Murcia and the University of Porto. 

The long series of experiments involving the synthesis of vinblastine analogues by the Polonovski coupling of catharanthine Nb-oxide derivatives with vindoline has culminated in the synthesis of vinblastine (290) itself, and this forms a fitting climax to any report on the past year s progress in indole alkaloid chemistry. 

Catharanthine N-oxide [2] was treated with vindoline in methylene chloride-trifluoroacetic anhydride at -50 5 coupling occurred, to give the immonium ion [3] which w is educed with sodium borohydride to provide the -deoxyvinblastine (anhydrovinblastine)... 

Earlier results in a study of the clinically important dimeric alkaloid vincaleukoblastine (122) had provided some evidence on the course of biosynthesis. The obvious correlation of (122) with vindoline (111) and catharanthine (112) has not been supported, owing to insignificant incorporations into (122) of the two monomeric bases. Using apical cuttings of C. roseus, however, where catabolic turnover of (111) and (112) is lower than in intact plants, the specific incorporation of (111) and (112) was observed. The level of incorporation was low, thus casting some doubt on whether vincaleukoblastine (122) is formed by dimerization of (111) and (112). 

The Polonovski reaction of demethoxycarbonylcatharanthine -oxide with trifluoroacetic anhydride in the presence of vindoline leads to a coupling reaction in which 16 -demethoxycarbonylanhydrovinblastine and its C-16 epimer are produced a third product was formulated as demethoxycarbonyl-catharan-thine with a 10-vindolinyl unit attached to position 3. A re-examination of this last product has shown that it is entirely analogous to that produced in the similar coupling of catharanthine iV-oxide with vindoline, and has the structure (251). 

C46HjgN40 Mr 810.99, cryst., mp. 216°C, [alp +42 (CHCI3) a dimeric indole alkaloid from Catharanthus roseus (see Vinca alkaloids), composed of two parts vindoline and catharanthine with a 10,3 -linkage. V. is a cytostatically active trace alkaloid and is accompanied by demethylated, deformylated, and deacety-lated alkaloids. V. also occurs in other Catharanthus species such as C. ovalis. C. longifolius, C. trichophyl-lus. It is one of the most important monoterpenoid indole alkaloids. 

Boger et al. have reported a single-step biomimetic coupling of catharanthine (124) with vindoline (123) to yield vinblastine (117) efficiently. Thus, the FeCls (5 equiv)-mediated coupHng of vindoline (123) and catharanthine (124) in 0.1 N FICl—CF3CH2OH is followed by the addition of the reaction mixture to a Fc2(ox)3 (iron(III) oxalate hexahydrate, 10 equiv) solution cooling to 0 °C and saturation with air. The subsequent addition of NaBFl4 (20 equiv) initiates both the reduction of the intermediate imi-... 

Two new analogues of catharanthine, 753 and 754, differing from catharanthine in the fusion of the indole ring to the non-aromatic portion of the iboga skeleton have been synthesized in racemic form and their reactivity toward coupling with vindoline examined. The [2,1] fused analogues (e.g., 753) were found to give low... 

Fig. 34.3 Total ion chromatogram (a) and extracted ion chromatogram (b) of a standard mixture solution, and extracted ion chromatogram (c) of Catharanthus roseus extract by CE-MS. CE conditions capillary, 65 cm length and 50 pm i.d, buffer 20 mM s ammonium acetate aqueous with 1.5 % acetic acid, temperature room temperature, sample, 50 mbar for 5 s injection MS-conditions ESI positive, sheath gas 4 psi dry gas 6 L/min dry temperature 130 °C capillary voltage 3.5 kV sheath liquid methanol/water = 1/1, with 0.1 % acetic acid flow rate of sheath liquid 4 pL/min. Peak identification 1 vinblastine 2 catharanthine 3 vindoline (From Ref. 8)... 

Extensive biotransformation studies have been conducted with the As-pidosperma alkaloid vindoline, but much less work has been done with monomeric Iboga and dimeric alkaloids from this plant. The long-standing interest in this group of compounds stems from the clinical importance of the dimeric alkaloids vincristine and vinblastine, both of which have been used for more than 2 decades in the treatment of cancer. Few mammalian metabolites of dimeric Catharanthus alkaloids have been characterized. Thus the potential role of alkaloid metabolism in mechanism of action or dose-limiting toxicities remains unknown. The fact that little information existed about the metabolic fate of representative Aspidosperma and Iboga alkaloids and Vinca dimers prompted detailed microbial, mammalian enzymatic, and chemical studies with such compounds as vindoline, cleavamine, catharanthine, and their derivatives. Patterns of metabolism observed with the monomeric alkaloids would be expected to occur with the dimeric compounds. 

Goodbody and co-workers (7/9) have examined the production of alkaloids in root and shoot cultures induced from seedlings of C. roseus. The pattern of alkaloids in the root cultures was similar to that of the roots from intact plants. Thus ajmalicine (39) and catharanthine (4) were produced, but no vindoline (3), a major leaf alkaloid, and no bisindole alkaloids. Similarly, the pattern of the alkaloid content of the shoot cultures was like that of the leaves of the intact plant, showing the presence of vindoline (3), catharanthine (4), and ajmalicine (39), with 3 predominating. A search for the bisindole alkaloids in the cultures indicated the presence of anhydrovinblastine (8) and leurosine (11) in the shoot cultures (2.6 and 0.3 xg/g fresh weight, respectively), but no vinblastine (1) or vincristine (2). 

Scott and co-workers have also reported on the isolation of alkaloids from C. roseus cell suspension cultures 126). The cell line used, identified as CRW, afforded akuammicine (49), catharanthine (4), and strictosi-dine (33), and feeding experiments with labeled tryptophan led to incorporation into ajmalicine (39), akuammicine (49), catharanthine (4), and vindoline (3). The ability to produce alkaloids was carried through 8 successive generations. 

It is well established that the iridoids are derived from two units of mevalonic acid (97), which itself is derived from acetyl-CoA. Mevalonate is also known to be a metabolic product of leucine (172), and the latter is a precursor of the monoterpene linalool (173). Wigfield and Wen (174) pursued the incorporation of leucine into the monoterpene unit in both vindoline (3) and catharanthine (4), where levels of 0.07 and 0.02%, respectively, were found, irrespective of the amount of precursor fed. This was important because, although initial results were obtained with [2- C] leucine, the specificity of incorporation was determined with 2- C-la-beled precursor. Two carbons in vindoline (3), C-8 and C-24, were en-... 

In vivo feeding experiments with singly and doubly labeled strictosidine (33) in C. roseus shoots afforded labeled ajmalicine (39), serpentine (40), vindoline (3), and catharanthine (4). Vincoside (85, page 37) was not incorporated into the alkaloids, suggesting that it was biologically inert 188). Brown and co-workers 190) conducted somewhat parallel studies examining the precursor relationship of strictosidine in C. roseus. Incorporation into tetrahydroalstonine (75), ajmalicine (39), catharanthine (4), and vindoline (3) was observed. 

The enzyme-catalyzed formation of anhydrovinblastine (8) from catharanthine (4) and vindoline (3) was first examined by Kutney and co-workers (170,219) using a cell-free preparation. [ao f- H]Catharanthine (4) and [acety/- C]vindoline (3) were incubated for 3-8 hr, both separately and jointly with a preparation from C. roseus, which led to the isolation of labeled anhydrovinblastine (8) and leurosine (11) incorporations were of the order of 0.54 and 0.36%, respectively. On this basis, anhydrovinblastine (8) was proposed as the key biosynthetic intermediate en route to vinblastine (1) and vincristine (2). 

FMN. Similar conversion yields for each of the isozymes were in the range 34-50%. Paralleling these data was the observation that horseradish peroxidase was also capable of converting vindoline (3) and catharanthine (4) to anhydrovinblastine (8) with the correct C-18 stereochemistry (222). 

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